Bacterial mosouito larva-killing agent

ABSTRACT

THE PRESENT BACTERIAL MOSQUITO LARVA-KILLING AGENT COMPRISES SPORES OF BACILLUS CEREUS VAR. JUROI ATCC 21281 AS AN ACTIVE INGREDIENT AND HAS A POSITIVE TOXIC EFFECT ONLY ON MOSQUITO LARVAE AND IS QUITE NON-TOXIC TO HUMANS, CATTLE AND FISH. THE SUFFICIENTLY EFFECTIVE SPORE CONCENTRATION OF THE MOSQUITO LARVA-KILLING AGENT IS 10**10 TO 10**11 SPORES PER GRAM OF THE AGENT, AND THE AGENT CAN BE USED DIRECTLY OR IN COMBINATIONS WITH A FILLER, EMULSIFIER, OR OLEAGINOUS LIQUID IN ANY FORM SUCH AS POWDERS, TABLETS CAPSULES OR AQUEOUS EMULSIONS.

United States Patent 3,651,215 BACTERIAL MOSQUITO LARVA-KILLING AGENT Satohiro Ibuki and Nobuo Fujiyoshi, Saitama-ken, Japan,

assignors to Juro Morita, Tokyo, Japan No Drawing. Continuation-impart of application Ser. No.

620,268, Mar. 3, 1967. This application Aug. 12, 1968,

Ser. No. 751,750

Int. Cl. A01n /00 U.S. Cl. 424-93 8 Claims ABSTRACT OF THE DISCLOSURE This application is a continuation-in-part of copending application Ser. No. 620,268, filed on Mar. 3, 1967, now abandoned.

This invention relates to a bacterial mosquito larvakilling agent, more particularly, a bacterial mosquito larva-killing agent showing a positive toxic effect on mosquito-larvae and yet being quite non-toxic to humans, cattle and fish.

Chemical insecticides have been so far used to kill such sanitary insect pests as flies and mosquitoes. However, such chemical insecticides not only elevate the resistance of insect pests to the insecticides until it is so high as to reduce the effect of the insecticides, but can also be very detrimental to humans, cattle, fish and useful insects. Therefore, the use of such insecticides has become increasingly suspect. Thus there has been recently, and it is to be expected that there will be in the future, much research on the utilization of natural enemies to take the place of conventional chemical insecticides. In Europe and America microbial insecticides composed mostly of Bacillus thuringiensis and showing a toxic etfect on general agricultural and forest insect pests have been used in practice. (For example, see A. Krieg: Bacillus Ihuringiensis Berliner, Berlin, 1961.) However, no great effect on sanitary insect pests can be expected from the natural enemies of agricultural and forest insect pests. Now, as a result of our hard efforts for the selection of bacteria which are non-toxic to humans, cattle, poultry and fish and yet which show a high and long-lasting toxic effect selectively on sanitary insect pests, the present inventors have succeeded in isolating Bacillus cereus var. juroi ATCC 21281 showing a selective killing effect upon mosquito larvae from farm soils 'in Iruma-gun, Saitama Prefecture, Japan, and have invented a novel bacterial insecticide for killing mosquito larvae.

Bacteriological properties of Bacillus cereus var. iuroi ATCC No. 21281 are given below:

Gram positive, aerobic sporeformer, motile. Actylmethylcarbinol produced. Acid from glucose.

3,651,215 Patented Mar. 21, 1972 No acid from arabinose, xylose, lactose and mannitol. No acid from saccharose and glycerol.

Lecithinase not produced.

Citrates utilization variable.

Pathogenic for mosquito larvae;

Not toxic to mice.

FORM

Rods of 0.7 x 2.5-2.7 microns.

The sporangia are not definitely swollen.

The spores are ellipsoidal, 0.4 x 2.0 microns (paracentral).

Comparison of the bacteriological properties of Bacillus cereus var. juroi ATCC No. 21281 with other microorganisms belonging to the genus Bacillus described in Bergeys Mannual of Determinative Bacteriology, 7th edition, etc., reveals its similarity to Bacillus cereus, as shown in Table 1.

TABLE 1 Bacillus sp I II III IV V B. B. i 1 1g: i- B: slearothermop u B. circulans. B. B. g. :l:

B. 111 B. coagulans.-- :1: :l: -p z/ u u- B. alvei Bacillus cereus var. juroi ATCC 21281.-- :1:

I: Acetylmethylcarbinol produced.

II: Citrates utilized.

III: Acid from arabinose and xylose.

IV: Acid from mannitol. V: Nitntes produced from nitrates (:1: shows a variability).

The species of the present microorganism is morphologically, biochemically similar to Bacillus cereus and thus has been determined to belong to the species Bacillus cereus. It is found that the present microorganism has a peculiar pathogenic nature for mosquito larvae, as shown in Table 2. Further, it is found that there are slight differ ences in its bacteriological properties, as shown in Table 3. Thus, the present microorganism was deemed a new variant species of Bacillus cereus and accordingly named Bacillus cereus var. iuroi.

8 SCC: Seibu Culture Collection. b 1AM: Institute of Applied Microbiology, Tokyo Univ. Concentration of spores: 5x10 spores/m1. of larval medium. 7

"' TABLE 3 Bacillus cereus Bacillus var. juroi Item cereus ATCC 21281 Physiological properties Saccharose utilization Glycerol utilization Lecithinase reaction Citric acid utilization i= Pathogenlcity Pathogenicity on mosquito larvae Relation between the toxicity on mosquito larvae and concentration of the present Bacillus cereus var. juroi ATCC 21281 is given in Table 4.

TABLE 4 Concentration of Number of Number 01 Number of Percent spores per ml. of inoculated produced produced effectivelarval medium larvae pupae adults ness N owns: (1; Sample mosquitoes: C'ulez pipieus Coquillett.

(2 Number of eating days: 2 days.

As shown in Table 4, it is desirable that the concentration of spores per m1. of the larval medium is about 5X spores, or more.

It is necessary in the present invention to make the mosquito larvae eat the present bacterial insecticide.

The toxic substance of the bacterial insecticide of the present invention on mosquito larvae is not yet completely clear. However, it is considered to be due to bacteria (endotoxin) from the fact that, when young larvae of mosquitoes eat the bacterial spores of this insecticide for only 2 days, substantially all of them will die. This toxin in the bacteria is characteristic of Bacillus cereus var. juroi ATCC 21281 according to the present invention.

In order to exemplify it, the toxicity on mosquito larvae of Bacillus cereus var. juroi ATCC 21281 according to the present invention is compared with toxicity of the typical insect pathogenic bacteria, Bacillus thuringiensis and fly pathogenic bacteria, Bacillus moritai ATCC 21282 in the following Table 5.

I Sample mosquitoes: C'ulea: pipiem Coquillett.

b U.S. patent coperrding application Ser. No. 753,874.

The results of tests of Bacillus thurinqicusir are taken from J. Insect. PathoL, 2, pp. 306 (1960).

The present invention is to provide a bacterial insecticide comprising spores of Bacillus cereus var. juror ATCC 21281 as an active ingredient.

The spores of Bacillus cereus var. iuroi ATCC 21281 are obtained in the following manner: Propagation, culturing and spore formation of the present bacteria are carried out in a culturing medium containing a suilicient carbon source such as molasses, date or glucose nitrogen source such as corn steep liquor, meat extract or peptone, and growth factors such as biotin or thiamine and being appropriate for spore formation by the ordinary aerated, submerged culturing method. a a r i The culturing is carried out at 20-35 0., preferably 27 C., and at a starting pH of 7-8 for 25-30 hours. The final whole culture is dried by spray or freezing-vacuum after centrifugal separation by means of a 'Sharples centrifuge or the like, or not, and further dried and properly pulverized to obtain-a stability of spore activity, whereby the dried powders of bacteria consisting mainly of spores or final whole culture containing the spores can be obtained.

the places where the larvae live. Furthermore a filler can be added to the thus obtained dry powders of-bacteria consisting mainly of spores or final whole culture. A sum,- ciently effective spore concentration of the product'insecticide is 10 to 10 spores per gram. 7

The filler itself, that is, powders of inactive substance; is well known and is not novel as an ingredient for the present bacterial insecticide. As ,the powders of inactive substances, such mineral'powders as bentonite, kaolin, talc, zeolite, alumina, diatomaceous earth, calcium carbonate, marble powder or lime powder, or volcanic ashes can be added to the dry powders of bacteria consisting mainly o sporesor final whole culture. 1 I v In preparing the insecticide by mixing the .dr'y powders of bacteria consisting mainly of spores or final whole culture with the filler, the insecticide can be offered in various forms such as aqueous emulsion, tablets, capsules or othe forms in addition to the powdery form.

In the case of an encapsulated insecticide, the dry pow-. ders of bacteria consisting mainly of spores are encased, in water-soluble capsules. A simple killing method based on spraying the capsuled insecticides at the places where the mosquito larvae. live is possible also in that case.

In the case of an emulsified-insecticide, the ordinary oleaginous liquid such as xylene,.kerosene, corn oil or whale oil, and such emulsifying agents as anionic surface active agents, i.e. salts of higher alcohol sulfate ester and alkylaryl-sulfonates; cationic surface active agents, i.e. aminoethylimidazolines and 'quatemary ammonium salts;

and non-ionic surface active agents, i.e. polyoxyethylene can be expected in addition to the effect of the present active ingredient.

EXAMPLE I 7 l f Bacillus cereus var. juroi ATCC 21281 is inoculated in a bouillon medium containing 1.0% meat extract, 1.0% peptone and 0.2% sodium chloride and having pH of 7.5 and culturing is conducted at 27 C. for 3O hoursrThen, the cultured medium is centrifuged, whereby the sporeformed cells can be obtained in a slurry state. The thus obtained cells are dried and pulverized, whereby the dry powders of bacteria consisting mainly of spores are obtained.

A powdery insecticide is prepared by mixing one part by weight of dry powders of bacteria consisting mainly of spores and 99 parts by weight of kaolin powders (dry basis). The thus prepared insectide contains 2.3)(10 spores per gram of the product insecticide. p

a medium containing 1.0% molasses, 1.0% corn steepv liquor, 0.1% 'KH3PO4 and 0.05% MgSO -7H O and hav- 1 ing a pH of 7.5, and .culturing is conducted tit-27 C. for

30 hours. The final whole cu1ture containing spores is spray-dried with hot air of 60 Q, whereby *dry powders of final whole culture are obtained.

A powdery insecticide is prepared by mixing two parts" by weight of dry powders of final whole culture and 98 parts by weight of diatomaceous earth (dry basis). The

6 thus prepared insecticide contains 8.4 10 spores per 2. A bacterial mosquito larva-killing agent according gram of the product insecticide. to claim 1 wherein 10 to 10 spores are contained in a gram of the killing agent.

EXAMPLE 3 3. A bacterlal mosquito larva-killmg agent accordlng An aqueous Qmlllslfied msectlclde 1S P p y II1 1X1I1g 5 to claim 1, wherein the carrier is a mineral powder. 10 Parts y Welght f y PQ of bacteria consisting 4. A bacterial mosquito larva-killing agent according mainly 0f SPURS P p 1n f p 10 p y to claim 1, wherein the carrier is an oleaginous liquid. Welght 0f Xylene, P y Welght Of all emulslfief 5. A bacterial mosquito larva-killing agent according fi a trademark of Surfadant Produced y Atlas to claim 1, wherein the mixture is in a capsuled form. Po e C and Parts by Weight of Water- 6. A bacterial mosquito larva-killing agent according The thus P p emulsified insecticide contains X to claim 1, wherein the mixture is in a tablet form. Spores P 1 and is used after being diluted to 5 t0 7. A bacterial mosquito larva-killing agent according 10 times the Original Volume 'Wlth Watefto claim 1, wherein said carrier is an inactive substance The bacterial mosquito larva-killing agents P p in selected from the group consisting of bentonite, kaolin,

foregoing Examples 1, 2 and 3 have the killing effects, 5 talc, zeolite, alumina, diatomaceous earth, calcium caras shown in Table 6.

TABLE 6 D 108 351? 5i mosquito Number of Number of Number of Percent larvae inoculated produced produced efiectivemedium larvae pupae adults ness Example 1 0.1 g 0 0 0 100 Example 2 0.5 g 50 0 0 100 Examp1e3 1 ml 50 0 0 100 Control 0 50 48 46 Note: (1) Sample mosquitoes: Oulea: pim'aes Coquillett.

(2) Tests on killing elfect only of the corresponding fillers of said Examples, which are free of the active ingredient, all show negative results.

The examples exemplified above are merely for illusbonate, marble powder, lime powder and volcanic ashes. tration of the present invention, and the present inven- 8. A method for killing mosquito larvae which comtion is not limited thereto. prises an effective mosquito larva-killing amount of spores The essential feature of the present invention is to use of Bacillus cereus var. juroi ATCC 21281 to said mosthe spores of Bacillus cereus var. juroi ATCC 21281 as quito larvae. an effective ingredient for killing mosquito larvae, singly Reference Cited or in combination with a suitable filler or carrier. Thus, UNITED STATES PATENTS any carrier can-be used so long as the carrier will not perish or inactivate the activity of the spores. 8/1967 Page 424 '93 Further, it is also possible to simultaneously use a 40 3711243 9/1966 Cords et 424-93 chemical insecticide or an attractant for egg-laying which OTHER REFERENCES ggi zg g z figf the the SPmes- Stephens: Canada, J. 2001., 30, 3040 1952 Heimpel et al.: Bacteriological Reviews, 24 (3), 266- 1. A bacterial mosquito larva-killing agent comprising 2 an efiective mosquito larva-killing amount of spores of September 1960' Bacillus cereus var. juroi ATCC 21281 as an active in- JEROME D. GOLDBERG Primary Examiner gredient and a carrier. 

